Small-molecule modulators of melanin expression

ABSTRACT

The present invention relates to small molecule modulators of melanin expression and methods of making the small molecules. Also disclosed are methods of increasing pigmentation in a cell which involve providing compounds of the present invention and contacting a cell with the compounds under conditions effective to induce melanin expression in the cell, thereby increasing pigmentation. The present invention also relates to compositions containing compounds of the present invention and a carrier.

This application is a division of U.S. patent application Ser. No.12/593,673, which is a national stage application under 35 U.S.C. §371of PCT/US2008/058716, filed Mar. 28, 2008, which claims the prioritybenefit of U.S. Provisional Patent Application Ser. No. 60/909,264,filed Mar. 30, 2007, all of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to small-molecule modulators of melaninexpression, compositions containing the compounds, and methods of usingthe compounds to induce melanin expression (i.e., in melanocytes) andincrease skin pigmentation.

The subject matter of this application was made with support from theUnited States Government under NIH Grant No. T32 AR007472. The U.S.Government has certain rights.

BACKGROUND OF THE INVENTION

Melanin is a complex biopolymer that is synthesized in organelles calledmelanosomes that are a unique feature of melanocytes. Melanocytes arecells that reside in the basal layer of the epidermis and transfermelanin to neighboring keratinocytes. In melanin synthesis, earlyoxidative steps in which phenolic and catecholic precursors are oxidizedto orthoquinones occurs. Regulation of melanin production occurs atmultiple points that primarily involve, but are not exclusive to, theregulation of tyrosinase, which is the key enzyme in melanin synthesis(Wang et al., “Tyrosinase Maturation Through the Mammalian SecretoryPathway: Bringing Color to Life,” Pigment Cell Res. 19:3-18 (2006); Wanget al., “Regulation of Tyrosinase Trafficking and Processing byPresenilins: Partial Loss of Function by Familial Alzheimer's DiseaseMutation,” Proc. Natl. Acad. Sci. USA 103:353-8 (2006)). However,because over 100 genes are implicated in control of pigmentation, otherregulatory targets are likely to exist.

Melanin is important for disease prevention, because it absorbsultraviolet light in the UVB and UVA spectrum. This protectskeratinocytes from the mutagenic effects of sunlight. In addition,melanin has a social function, because it determines skin and haircolor. Cutaneous cancers such as squamous cell carcinoma and basal cellcarcinoma are directly linked to exposure to ultraviolet irradiation,and it is widely accepted that people with light skin are at much higherrisk to develop skin cancer due to a comparative lack of melanincompared with dark skinned individuals (Gallagher et al., “AdverseEffects of Ultraviolet Radiation: A Brief Review,” Prog. Biophys. Mol.Biol. 92(1):119-31 (2006)).

Agents that increase melanin synthesis are of interest, because of theirpotential use as “natural sunscreens” that stimulate the skin's residentmelanocytes to produce and transfer more melanin to epidermalkeratinocytes, thus decreasing the incidence of skin cancer. There is asignificant need to identify new compounds with improved ability toinduce melanin production and transfer. The development of libraries ofcompounds that can be rapidly screened for their ability to modulatemelanin production would facilitate drug discovery that could have aprofound impact on the incidence of skin cancer.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a compound formed bya reaction of a carboxylic acid and an amine. The carboxylic acid isselected from

and the amine is selected from

A second aspect of the present invention relates to a compound accordingto formula (I)

wherein,

-   -   R¹ is selected from H and straight or branched chain C₁ to C₆        hydrocarbon;    -   R² is selected from —O—R³, —NH—R³, and —S—R³, where R³ is        selected from H, straight or branched chain C₁ to C₆        hydrocarbon, and alkoxyalkyl;    -   W is a straight or branched chain C₁ to C₆ hydrocarbon,        —(CH₂)_(p)NH—, or —(CH₂)_(p)N(CH₃)—, where p is an integer from        0 to 3;    -   X is selected from (CH₂)_(n), O, —(CH₂)_(q)NH—, or        —(CH₂)_(q)N(CH₃)—, where n is an integer from 0 to 8 and q is an        integer from 0 to 3;    -   Y is selected from (CH₂)_(m), where m is an integer from 0 to 3;        and    -   Z is selected from

where Q is selected from H and halogen.

A third aspect of the present invention relates to a compound accordingto formula (II)

wherein,

-   -   R⁴ is selected from H and straight or branched chain C₁ to C₆        hydrocarbon;    -   Y is selected from O, N—R⁵, and S—R⁵, where R⁵ is selected from        H and straight or branched chain C₁ to C₆ hydrocarbon;    -   X is selected from R⁶, O—R⁶, NH—R⁶, NR⁶ ₂,

wherein each R⁶ is independently selected from H, straight or branchedchain C₁ to C₆ hydrocarbon, and halogen; q is an integer from 0 to 5;and Z is selected from H, halogen, and C₁ to C₃ hydrocarbon.

A fourth aspect of the present invention relates to a method ofincreasing pigmentation in a cell. This method involves providing acompound according to the first, second, or third aspects of the presentinvention, or a combination thereof, and contacting a cell with thecompound under conditions effective to induce melanin expression in thecell, thereby increasing pigmentation.

A fifth aspect of the present invention relates to a compositioncontaining a carrier, and a compound according to the first, second, orthird aspects of the present invention, or combinations thereof.

A sixth aspect of the present invention relates to a method ofincreasing skin pigmentation. This method involves applying acomposition according to the fifth aspect of the present invention toskin on a subject in a manner and quantity effective to induce melaninproduction in contacted skin cells, thereby increasing skin pigmentationwithin a treated area of the skin.

A seventh aspect of the present invention relates to a method of makingan amide compound according to the first, second, or third aspects ofthe present invention. This method involves providing a carboxylic acidand reacting the carboxylic acid with an amine under conditionseffective to make an amide compound according to the first, second, orthird aspects of the present invention.

An eighth aspect of the present invention relates to a method of makinga compound according to formula (I) where X is oxygen. This methodinvolves providing an isoquinoline carboxylic acid according to formula(III)

wherein R¹, R², and W are defined as in formula (I); and reacting theisoquinoline carboxylic acid of formula (III) with an alcohol of formula(IV)

HO-(y)-(z)  (IV),

wherein Y and Z are defined as in formula (I). The reaction is carriedout under conditions effective to make a compound according to formula(I) where X is oxygen.

A ninth aspect of the present invention relates to a method of making acompound of formula (I) where X is (CH₂)_(n). This method involvesproviding an isoquinoline carboxylic acid according to formula (III)above. The isoquinoline carboxylic acid according to formula (III) isconverted to a Weinreb amide via treatment with carbonyl diimidazolefollowed by N,O-Dimethylhydroxylamine. The resulting Weinreb amide isthen treated with an alkyl Grignard reagent of formula (V)

(Z)—(Y)—(CH₂)_(n)—MgBr,  (V)

where Y is selected from (CH₂)_(m), where m is an integer from 0 to 3, Zis selected from

and Q is selected from H and halogen. These steps are carried out underconditions effective to make a compound of formula (I) where X is(CH₂)_(n).

A tenth aspect of the present invention relates to a method of making acompound of formula (II) having an ester linkage in the sidechain. Thismethod involves providing a carboxylic acid according to formula (VII)

where R⁴ and Y are defined as in formula II, and reacting the carboxylicacid of formula (VII) with an alcohol of formula (VIII)

R⁶—OH  (VIII)

where R⁶ is a straight or branched chain C₁ to C₆ hydrocarbon, underconditions effective to make the compound of formula (II).

An eleventh aspect of the present invention relates to a method ofmaking a compound of formula (II) where X is R⁶, a straight or branchedchain C₁ to C₆ hydrocarbon. This method involves providing a carboxylicacid according to formula (VII)

where R⁴ and Y are defined as in formula (II), and converting thecarboxylic acid to a Weinreb amide via treatment with carbonyldiimidazole followed by N,O-Dimethylhydroxylamine. The Weinreb amide istreated with an alkyl Grignard reagent, (R⁶)-MgBr under conditionseffective to make a compound of formula (II) where X is a hydrocarbon asdefined above.

A twelfth aspect of the present invention is directed to a method ofmaking an ester compound according to formula (II). This method involvesproviding an acyl halide according to formula (IX)

where A is halogen and R⁴ and Y are as defined in formula (II), andreacting the acyl halide with an alcohol of formula (VIII)

R⁶—OH  (VIII)

where R⁶ is a straight or branched chain C₁ to C₆ hydrocarbon, underconditions effective to make the compound of formula (II).

Several compounds of the present invention have been shown to induceincreased melanin production in treated melanocytes, and importantlythis occurs without concomitant increased cellular proliferation by thetreated melanocytes. As a consequence, the compounds of the presentinvention, and compounds derived therefrom, are expected to be useful intopical compositions for the treatment of skin to induce increased skinpigmentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing how a small combinatoriallibrary of 75 low molecular weight (<500 AMU), structurally diverseheterocyclic compounds was synthesized.

FIG. 2 is a graph showing melanin quantity and cell number (±S.D.) upon2.5μM small molecule treatment normalized to DMSO vehicle control.

FIG. 3 is a pair of brightfield images of melan-a melanocytes culturedin the presence and absence of 2.5μM of compound A7.

FIG. 4 is a graph showing dose response of melanin quantity and cellnumber (±S.D.) upon A7 treatment normalized to DMSO vehicle control.

FIG. 5 shows images and a graph of hematoxylin stained melan-amelanocytes cultured in the presence and absence of 2.5μM of compoundA7.

FIG. 6 is a graph showing tyrosinase assay (±S.D.) of melan-amelanocytes cultured in the presence and absence of 2.5μM melanogenesisinducing small molecules.

FIG. 7 is an image of a two-dimensional differential in gelelectrophoresis. In total, 24 unique protein spots were reproduciblyincreased or decreased in two 2D-DIGE analyses upon treatment with A7 ascompared to DMSO control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification of novel compoundsthat are capable of inducing melanocyte expression of melanin, andtherefore are capable of use for increasing skin pigmentation (i.e., inkeratinocytes). The compounds, compositions suitable for administeringthem, and their methods of use are described herein.

A first aspect of the present invention relates to a compound formed bya reaction of a carboxylic acid and an amine. The carboxylic acid isselected from

and the amine is selected from

The reaction of a carboxylic acid with an amine to form an amide is acommon reaction known by those of ordinary skill in the art. Typically,the carboxylic acid is activated by reacting it with an activatingagent. For example, the carboxylic acid may be transformed to an acidchloride by treatment with oxalyl chloride or another similar agent. Theacid chloride is then reacted with the amine in the presence of a baseto form the amide.

Compounds of the present invention formed by a reaction of a carboxylicacid and an amine may have any combination of a carboxylic acid selectedfrom the above “library” of carboxylic acids and an amine selected fromthe above “library” of amines. Examples of particular compounds of thepresent invention formed by a reaction of a carboxylic acid and an amineare set forth in Table 1.

TABLE 1 Compounds Formed by a Reaction of a Carboxylic Acid and an AmineCom- pound Name Structure A5  N-(4- fluorophenethyl)- 2-ethylquinoline-3-carboxamide

A7  N-(3- fluorophenethyl)- 2-ethylquinoline- 3-carboxamide

C1  N-((furan-2-yl) methyl)furan-2- carboxamide

C2  N-butylfuran- 2-carboxamide

C5  N-(4- fluorophenethyl) furan-2- carboxamide

C7  N-(3- fluorophenethyl) furan-2- carboxamide

C10 (furan-2-yl) (2,2,6,6- tetramethylpiperidin- 1-yl)methanone

D10 (2,2,6,6- tetramethylpiperidin- 1-yl) (perfluorophenyl) methanone

D12 2,3,4,5,6- pentafluoro-N-(5- methylisoxazol-3- yl)benzamide

E4  (benzo[1,3]dioxol- 5-yl)(4-(4- fluorophenyl) piperazin-1-yl)methanone

By virtue of the activity of the quinoline compounds identified in Table1 above, the present invention also relates to compounds according toformula (I)

wherein,

-   -   R¹ is selected from H and straight or branched chain C₁ to C₆        hydrocarbon, preferably hydrogen, methyl, ethyl, propyl,        isopropyl, or t-butyl;    -   R² is selected from —O—R³, —NH—R³, and —S—R³, where R³ is        selected from H, straight or branched chain C₁ to C₆        hydrocarbon, and alkoxyalkyl, preferably methoxy, ethoxy,        propoxy, methoxyethyl, N-methyl amino, N-ethyl amino, N-propyl        amino, methylthio, ethylthio, or propylthio;    -   W is optional and can be a straight or branched chain C₁ to C₆        hydrocarbon, —(CH₂)_(p)NH—, or —(CH₂)_(p)N(CH₃)13 , where p is        an integer from 0 to 3, preferably 0 or 1;    -   X is selected from (CH₂)_(n), O, —(CH₂)_(q)NH—, or        —(CH₂)_(q)N(CH₃)—, where n is an integer from 0 to 8 and q is an        integer from 0 to 3, preferably 0 or 1;    -   Y is selected from (CH₂)_(m), where m is an integer from 0 to 3;        and    -   Z is selected from

where Q is selected from H and halogen.

As used herein, the straight or branched chain hydrocarbons can bealiphatic or non-aliphatic, and includes both saturated hydrocarbons,monounsaturated hydrocarbons, and polyunsaturated hydrocarbons. Thus,this term is intended to include alkylene groups that contain a singlecarbon and up to a defined upper limit, as well as alkenyl groups andalkynyl groups that contain two carbons up to the upper limit, whetherthe carbons are present in a single chain or a branched chain.

As used herein, halogens can be any one of fluorine, chlorine, bromine,and iodine. Of these, fluorine and chlorine are preferred.

Preferred compound according to formula (I) include compounds A5 and A7of Table 1.

Compounds of formula (I) having an ester group in the sidechain can beformed by standard esterification reactions between the appropriateisoquinoline carboxylic acid and an alcohol activated by reaction with,for example, dicyclohexyl carbodiimide. Accordingly, another aspect ofthe present invention relates to a method of making a compound offormula (I) where X is oxygen. This method involves providing anisoquinoline carboxylic acid according to formula (III)

wherein R¹, R², and W are defined as in formula (I); and reacting theisoquinoline carboxylic acid of formula (III) with an alcohol of formula(IV)

HO-(y)-(z)  (IV),

wherein Y and Z are defined as in formula (I). The reaction is carriedout under conditions effective to make a compound according to formula(I) where X is oxygen.

Alternatively, compounds of formula (I) having an ester in the sidechain (i.e., where X is O) can also be formed by using an acyl chlorideaccording to formula (VI)

plus an alcohol according to formula (IV).

Compounds of formula (I) having a ketone group in the side chain (i.e.,where X is (CH₂)_(n)) can also be formed. Accordingly, another aspect ofthe present invention relates to a method of making a compound offormula (I) where X is (CH₂)_(n). This method involves providing anisoquinoline carboxylic acid according to formula (III) above. Theisoquinoline carboxylic acid according to formula (III) is converted toa Weinreb amide via treatment with carbonyl diimidazole followed byN,O-Dimethylhydroxylamine according to the procedure of Nahm et al.,Tetrahedron Letters 22:3815-3818 (1981), which is hereby incorporated byreference in its entirety. The resulting Weinreb amide is then treatedwith an alkyl Grignard reagent of formula (V)

(Z)—(Y)—(CH₂)_(n)-MgBr,  (V)

according to common procedures known by those of ordinary skill in theart, where Z and Y are defined as according to formula (I). These stepsare carried out under conditions effective to make a compound of formula(I) where X is (CH₂)_(n).

Compounds of formula (I) having an amide group in the sidechain can beformed by standard peptide coupling reactions in the manner describedabove.

By virtue of the activity of the furan compounds identified above inTable 1, the present invention also relates to compounds according toformula (II)

wherein,

-   -   R⁴ is selected from H and straight or branched chain C₁ to C₆        hydrocarbon, preferably hydrogen, methyl, ethyl, or propyl;    -   Y is selected from O (e.g., furans), N—R⁵ (e.g., pyrroles) and        S—R⁵ (e.g., thiophenes), where R⁵ is selected from H and        straight or branched chain C₁ to C₆ hydrocarbon, preferably        hydrogen, methyl, ethyl, or propyl;    -   X is selected from R⁶, O—R⁶, NH—R⁶, NR⁶ ₂,

where each R⁶ is independently selected from H, straight or branchedchain C₁ to C₆ hydrocarbon, and halogen, preferably hydrogen, methyl,ethyl, propyl, fluorine, or chlorine; q is an integer from 0 to 5; andR⁷ is selected from H, halogen, and C₁ to C₃ hydrocarbon, preferablyhydrogen, fluoride, chlorine, methyl, ethyl, propyl, or isopropyl.

As used herein, the straight or branched chain hydrocarbons and halogensrecited in formula (II) can be defined as above for formula (I). Thesame hydrocarbons and halogens are also preferred in this embodiment.

Preferred compounds according to this aspect of the present inventionare compounds C1, C2, C5, C7, and C10 of Table 1.

Compounds of formula (II) having an amide group in the sidechain can beformed by standard peptide coupling reactions in the manner describedabove.

Compounds of formula (II) having an ester group in the sidechain can beformed by standard esterification reactions in the manner describedherein.

Compounds of formula (II) having a ketone group in the sidechain can beformed by a standard ketone-forming reaction in the manner describedherein.

The compounds of the present invention can also be in the form of asalt, preferably a pharmaceutically acceptable salt. The term“pharmaceutically acceptable salt” refers to those salts that retain thebiological effectiveness and properties of the free bases or free acids,which are not biologically or otherwise undesirable. The salts areformed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, N-acetylcysteine and the like. Other salts are known tothose of skill in the art and can readily be adapted for use inaccordance with the present invention.

As demonstrated in the examples, the compounds of the present inventionhave an ability to increase pigmentation in a cell (i.e., eitherdirectly in melanocytes or indirectly in keratinocytes). This method iscarried out by providing a compound of the present invention, preferablythough not exclusively those compounds identified in Table I or definedunder formulae (I) and (II), and then contacting a cell with thecompound under conditions effective to induce melanin expression in thecell, thereby increasing pigmentation.

Cells to be contacted in accordance with the present invention includeany cells responsible for producing melanin, specifically melanocytes.Because the compounds of the present invention have the ability toinduce melanin expression in melanocytes, and thereby increase melanincontent of keratinocytes, it is intended for the compounds to beadministered to skin in the form of an appropriate composition. Thecells are preferably contacted in vivo after application of thecomposition to the skin of a subject, preferably a human subject.

The compositions of the present invention are preferably formulated tobe administered by topical application to the skin (i.e., keratinoustissue). Accordingly, the composition preferably has good aestheticproperties and will not cause any safety or toxicity concerns.

The carrier used in this and other compositions of the present inventioncan be in a wide variety of forms, including emulsion carriers, such asoil-in-water, water-in-oil, and oil-in-water-in-silicone emulsions,creams, ointments, ophthalmic ointments, aqueous solution, lotions,gels, or aerosols. As will be understood by the skilled artisan, a givencomponent will distribute primarily into either the water oroil/silicone phase, depending upon the water solubility/dispersibilityof the component in question. A safe and effective amount of carrier isfrom about 50% to about 99.99%, preferably from about 80% to about99.99%, more preferably from about 90% to about 98%, and most preferablyfrom about 90% to about 95% of the composition.

Emulsions generally contain an effective amount of a compound of thepresent invention and a lipid or oil. Lipids and oils may be derivedfrom animals, plants, or petroleum, and can be natural or synthetic.Preferred emulsions also contain a humectant such as glycerin. Emulsionswill preferably further contain from about 1% to about 10%, morepreferably from about 2% to about 5%, of an emulsifier, based on theweight of the carriers. Emulsifiers may be ionic, anionic, or cationic.The emulsion may also contain an anti-foaming agent to minimize foamingupon application to the keratinous tissue. Anti-foaming agents includehigh molecular weight silicones and other materials well known in theart for such use.

Suitable emulsions may have a wide range of viscosities, depending uponthe product form. Exemplary low viscosity emulsions, which arepreferred, have a viscosity of about 50 centistokes or less, morepreferably about 10 centistokes or less, most preferably about 5centistokes or less. The emulsion may also contain anti-foaming agentsto minimize foaming upon application to the skin.

Other preferred carriers include oil-in-water emulsions having acontinuous aqueous phase and a hydrophobic, water-insoluble phasedispersed therein. Preferred oil-in-water emulsions comprise from about25% to about 98%, preferably from about 65% to about 95%, and morepreferably from about 70% to about 90% water by weight of the carrier.

The hydrophobic phase is dispersed in the continuous aqueous phase. Thehydrophobic phase may contain water insoluble or partially solublematerials such as are known in the art including, but not limited to,silicones. The compositions of the present invention include, but arenot limited to, lotions and creams, and may comprise a dermatologicallyacceptable emollient. As used herein, “emollient” refers to a materialuseful for preventing or relieving dryness, as well as for protectingthe skin. A wide variety of suitable emollients are known and may beused herein. Numerous examples of materials suitable for use as anemollient are provided in Sagarin, Cosmetics, Science, and Technology2nd Edition Vol. 1, pp 3243 (1972), which is hereby incorporated byreference in its entirety. A preferred emollient is glycerin. Glycerinis preferably used in an amount of from about 0.001% to about 20%, morepreferably from about 0.01% to about 10%, and most preferably from about0.1% to about 5%.

Lotions and creams according to the present invention generally comprisea solution carrier system and one or more emollients. Lotions typicallycomprise from about 1% to about 20%, preferably from about 5% to about20%, of emollient; from about 50% to about 90%, preferably from about60% to about 80%, water; and an effective amount of a compound of thepresent invention.

Ointments of the present invention may comprise a simple carrier base ofanimal or vegetable oil or semi-solid water soluble carriers. Ointmentsmay further comprise a thickening agent and/or an emollient. Forexample, an ointment may comprise from about 2% to about 20% of anemollient, about 0.1 to about 2% of a thickening agent, and an effectiveamount of a compound of the present invention. To enhance thepercutaneous absorption of the active ingredients, one or more of anumber of agents can be added to the topical formulations, including butnot limited to, dimethylsulfoxide (DMSO), dimethylacetamide,dimethylformamde, surfactants, azone, alcohol, acetone, propyleneglycol, and polyethylene glycol. In addition, physical methods can alsobe used to enhance transdermal penetration such as e.g., byiontophoresis or sonophoresis. Alternatively, or in addition, thecomposition may be delivered in liposomes.

Compositions according to the present invention may also includeoptional components, which should be suitable for application tokeratinous tissue, i.e., when incorporated into the composition they aresuitable for use in contact with human keratinous tissue without unduetoxicity, incompatibility, instability, allergic response, and the likewithin the scope of sound medical judgment. In addition, such optionalcomponents are useful provided that they do not unacceptably alter thebenefits of the active compounds of the present invention. The CTFACosmetic Ingredient Handbook, Second Edition (1992) (which is herebyincorporated by reference in its entirety), describes a wide variety ofnon-limiting cosmetic and pharmaceutical ingredients commonly used inthe skin care industry, which are suitable for use in the compositionsof the present invention. Examples of these ingredient classes includeabrasives, absorbents, aesthetic components such as fragrances,pigments, colorings, essential oils, skin sensates, astringents (e.g.,clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate,hazel distillate), anti-acne agents, anti-caking agents, antifoamingagents, antimicrobial agents, antioxidants, binders, biologicaladditives, buffering agents, bulking agents, chelating agents, chemicaladditives, colorants, cosmetic astringents cosmetic biocides,denaturants, drug astringents, external analgesics, film formers ormaterials such as polymers for aiding the film-forming properties andsubstantivity of the composition (e.g., copolymer of eicosene and vinylpyrrolidone), opacifying agents, pH adjusters, propellants, reducingagents, sequestrants and/or healing agents (e.g., panthenol andderivatives such as ethyl panthenol), aloe vera, pantothenic acid andits derivatives, allantoin, and bisabolol), skin treating agents,thickeners, and vitamins and derivatives thereof.

The compounds of the present invention stimulate melanin production andcan be used topically for darkening skin, or to produce a safe tan.Accordingly, another aspect of the present invention relates to a methodof increasing skin pigmentation. This method involves applying acomposition of the present invention to skin on a subject in a mannerand quantity effective to induce melanin production in contacted skincells, thereby increasing skin pigmentation within a treated area of theskin.

The compounds of the present invention are useful in treating skinconditions where insufficient skin pigmentation is produced, or wherethe subject, for cosmetic purposes, simply wishes to develop a “sunlesstan” or to augment tanning induced by a limited exposure to sunlight orultraviolet light. Unlike previously known indoor tanning compositions,the compounds of the present invention actually produce additionalmelanin in the skin, and thus protect the skin from ultravioletradiation.

The appropriate dose regimen, the amount of each dose administered, andspecific intervals between doses of the active compound will depend uponthe particular active compound employed, the conditions of the patientbeing treated, and the nature and severity of the disorder or conditionsbeing treated. Preferably, the active compound is administered in anamount and at an interval that results in the desired treatment of orimprovement in the disorder or condition being treated.

As one skilled in the art will readily appreciate, the compounds of thepresent invention can be used alone or in combination with one another,as well as in combination with the other melanin stimulating compoundsdescribed below, and with any other pigment affecting compounds.

The compounds of the present invention can be administered by any meansthat results in contact of the active agent with its site of action inthe body of a mammal. The compounds can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. Each can be administered alone, butis preferably administered with a carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The compositions of the present invention are also useful for cosmeticpurposes. Cosmetic applications include the topical application ofcompositions containing one or more compounds that affect pigmentationto enhance or otherwise alter the visual appearance of skin or hair.Occurrences in the skin or hair of noticeable but undesired pigmentationcan be treated using the methods of the present invention.

An effective dosage and treatment protocol can be determined byconventional means, starting with a low dose in laboratory animals, andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Animal studies,preferably mammalian studies, are commonly used to determine the maximaltolerable dose, or MTD, of a bioactive agent per kilogram weight. Thoseskilled in the art can extrapolate doses for efficacy and avoidance oftoxicity to other species, including humans.

Any one or a number of endpoints can be used to determine when to stoptreatment. For example, endpoints can be defined subjectively such as,for example, when the subject is simply “satisfied” with the results ofthe treatment. Alternatively, endpoints can be defined objectively. Forexample, the subject's skin or hair in the treated area can be comparedto a color chart. Treatment is terminated when the color of the skin orhair in the treated area is similar in appearance to a color chart.Alternatively, the reflectance of the treated skin or hair can bemeasured, and treatment can be terminated when the treated skin or hairattains a specified reflectance. In another method, the amount ofmelanin in the skin or hair can be measured. Melanin content can bedetermined in any way known in the art, including by histologicalmethods, with or without enhancement by stains for melanin.

Compositions of the present invention are preferably administeredpursuant to this aspect of the present invention by topical application.For topical administration, the compounds of the present invention canbe formulated as a foam or mousse, solution, gel, lotion, ointment,cream, suspension, paste, liniment, powder, tincture, aerosol,transdermal drug delivery system, or the like, in a pharmaceutically orcosmetically acceptable form by methods well known in the art. Thecomposition can be in any variety of forms common in the pharmaceuticalor domestic arts for topical application to animals or humans, includingsolutions lotions, sprays, creams, ointments, salves, gels, aerosols,etc., as set forth above. Preferred agents are those that are viscousenough to remain on the treated area, those that do not readilyevaporate, and/or those that are easily removed by rinsing with watertopically with the aid of soaps, cleansers, and/or shampoos. Actualmethods for preparing topical formulations are known or apparent tothose skilled in the art.

Although the formulations and compositions are preferably deliveredtopically, it is also contemplated that the compositions can bedelivered by various transdermal drug delivery systems, such astransdermal patches as known in the art.

EXAMPLES

The examples below are intended to exemplify the practice of the presentinvention but are by no means intended to limit the scope thereof.

Example 1 Synthesis and Analysis of a Library of Small Molecules

A 75 member combinatorial library was synthesized by reacting acidchlorides of carboxylic acids A-E with amines 1-15 (FIG. 1) (Dothager etal., J. Am. Chem. Soc. 127:8686 (2005), which is hereby incorporated byreference in its entirety). Carboxylic acid and amine components usedfor synthesizing the library were selected based on the presence ofindividual substructures in other “biologically active” compounds.Briefly, acid chlorides of A-E (1 eq) were prepared by reacting oxalylchloride (3 eq), DMF (0.01 eq) and A-E in dry CH₂Cl₂ for 18 hours.Solvent was then removed under reduced pressure. Triethylamine (1.05 eq)and acid chlorides were added to 1-15 (1.05 eq) in dry CH₂Cl₂. DMAP(0.05 eq) was then added resulting in vigorous evolution of gas, andreactions were stirred at RT for 36 hours. Crude reaction mixtures werepassed through a plug of silica gel, eluted with 1:1 hexanes:ethylacetate, and dried. RP-HPLC and ESI-MS confirmed all reactions yieldedpure desired products in varying yields. The purified library memberswere diluted to 25 mM stocks in DMSO for screening. All compounds of thelibrary were confirmed by HPLC and MS data.

Example 2 Melanin Induction by Small Molecules

The melan-a murine melanocyte cell line was chosen for libraryscreening. This is a non-tumorogenic line of melanocytes derived fromthe skin of C57B1/6J mice (Bennett et al., Int. J. Cancer 39:414 (1987),which is hereby incorporated by reference in its entirety). These cellsare ideal for phenotypic melanin screening as they proliferate quickly,are highly differentiated, and have been extensively studied (Snyder etal., Chemistry & Biology 12(4):477 (2005), which is hereby incorporatedby reference in its entirety). Melanocytes were cultured in RPMI 1640media supplemented with 10% FBS and 0.2 μM TPA, at 37° C. under 5% CO₂,95% atmosphere. For screening, 1×10⁵ melan-a melanocytes were plated in24 well tissue culture plates in a volume of 2 ml. After 18 hrincubation, 1 ml of media was removed and replaced by 1 ml media dopedwith small molecule, forskolin positive control, or DMSO vehicle controlfor 3 days. Initial screening was performed at a 2.5μM dose (0.01%DMSO). Cells were harvested by trypsinization, washed 2X with PBS (pH7.2), and counted by hemocytometry. Cell counts were used to assesstreatment effects on cell proliferation. Melanin production wasquantified by lysing cell pellets in 800 μl of 1 M NaOH for 2 hrs onice, vigorously resuspending the lysed material by pipette, reading theabsorbance at 490 nm of 200 μl aliquots of total lysate, and referencingto a standard synthetic melanin (Sigma) curve. The 75 member library wasscreened in triplicate. Melanin production was normalized to cell count(A₄₉₀/million cells) and fold change against DMSO was calculated (FIG.2).

Ten compounds from the library initially showed some activity forinducing melanin production. These included compounds A5, A7, C1, C2,C5, C7, C10, D10, D12, and E4 (see Table 1 above). Five library memberssignificantly induced (greater than 40% increase) visible melaninproduction (FIG. 3) with similar potency to forskolin (FIG. 2).Importantly, the selected compounds did not affect cellularproliferation. Interestingly, there is a preference for fluorinatedaromatic substituents. Importantly, compounds A5, A7, and C7, whichcontain the fluorinated aromatic substituents, were previously unknown.Subsequent analysis identified compound A7 as the most active hit, andstudies were performed to further characterize its activity. First, A7dose response was investigated (FIG. 4). Treatment with 2.5 μM A7provided the optimal melanogenic response. Importantly, there was nosignificant effect on cell proliferation at concentrations up to 5μM.

Example 3 Influence of Compound A7 on Melanocyte Dendricity

In addition to melanin production, A7 treatment induced changes in cellmorphology. Melanocytes are highly dendritic cells, and dendricity hasbeen correlated with melanin production (Kosano et al., Biochim.Biophys. Acta 1499:11 (2000); Scott et al., J. Invest. Dermatol. 126:855(2006), which are hereby incorporated by reference in their entirety).Dendricity was assessed by histochemical hematoxylin staining of melan-amelanocytes with and without 2.5 μM A7 treatment. 1×10⁴ melan-amelanocytes were cultured onto vitrogen coated cover slips and treatedwith 2.5 μM A7 or vehicle control for 72 hours. Cells were fixed in 3.7%formalin/PBS, stained with hematoxylin, and imaged. Dendrites per cellwere manually quantitated, and the percentage of cells with greater than2 dendrites was calculated. As seen in FIG. 5, A7 treatment increasesdendricity in addition to melanin formation: 36% of untreated and 69% ofA7 treated melan-a melanocytes had greater than 2 dendrites.

Example 4 Influence of Compound A7 on Tyrosinase Activity

To further understand the mode of action for A7, tyrosinase activity wasquantitated. Tyrosinase functions to hydroxylate tyrosine to dopaquinoneand L-DOPA in the first step of melanin production (Ito, Pigment CellRes. 16:230 (2003), which is hereby incorporated by reference in itsentirety). As such, increased tyrosinase activity leads to increasedmelanin production. Tyrosinase activity can be measured by monitoringthe production of tritiated water upon the hydroxylation ofL-tyrosine-3,5-[³H] to L-DOPA (Pomerantz, J. Biol. Chem. 241(1):161(1966); Park et al., J. Biol. Chem. 268:11742 (1993), which are herebyincorporated by reference in their entirety).

Similarly to library screening, in quadruplet, melanocytes were culturedwith and without 2.5μM small molecule treatment for 3 days. Resultingcell pellets were lysed in 75μl of 80 mM K₂PO₄, 1% CHAPS, 2 mM PMSF, andPICO2 protease inhibitor cocktail (CalBioChem) for 2 hours on ice.Lysates were then pelleted (10 minutes, 4° C., 10,000 rpm) and proteinconcentration was determined by Bradford assay. 2.5 μg aliquots of totalprotein were incubated in 250μl of 80 mM K₂PO₄ containing 50 nML-tyrosine, 25 nM L-DOPA, and 5μCi of L-tyrosine-3,5-[³H] for 60 minutesat 37° C. Proteins were precipitated (375μl 0.2% BSA and 375μl 10% TCA)and pelleted (10 minutes, 4° C., 10,000 rpm). 750 μl of the resultingsupernatent was added to 500μl of a 1:2 washed charcoal:80 mM K₂PO₄suspension, nutated for 1 hour, and charcoal was pelleted (15 minutes,4° C., 10,000 rpm). Aliqouts of 1.0 ml of the tritiated water containingsupernatant were transferred to scintillation vials containing 5.0 mlscintillation fluid, and ³H levels were counted. Denatured proteinsamples of boiled aliquots served as negative controls, and forskolinserved as a positive control. As seen in FIG. 6, A7 treatment increasestyrosinase activity in melanocyte culture, although to a lesser degreethan forskolin treatment. Additionally, Western blot analysis suggestscellular levels of tyrosinase are not altered.

Example 5 Protein Analysis of Melanocytes Treated with Compound A7

To identify changes in specific protein amounts in the control versus A7treated cells, two dimensional, differential gel electrophoresis wasemployed. In total, 24 unique protein spots were reproducibly increasedor decreased in two 2D-DIGE analyses upon treatment with A7 as comparedto DMSO control (FIG. 7). These proteins are in the process of beingidentified by MALDI-ToF mass spectrometry.

Discussion of Examples 1-5

In summary, phenotypic screening of a focused 75 member combinatoriallibrary was used to identify a number of novel small molecules capableof inducing melanogenesis in melanocytes in culture. Of these, compoundA7 proved to produce the best results. A7 has similar potency to theknown melanogenic inducing agent forskolin, and does not exhibitcytotoxic effects in cell culture. In addition to melanin formation, A7treatment results in increased cellular dendricity and tyrosinaseactivity, processes commonly associated with increased pigmentation.This novel compound represents a new class of melanogenesis inducingsmall molecules, structurally distinct from other compounds with similarfunction.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A compound formed by a reaction of a carboxylic acid and an amine,wherein the carboxylic acid is selected from:

and the amine is selected from:


2. The compound according to claim 1, wherein the compound is selectedfrom the group of N-(4-fluorophenethyl)-2-ethylquinoline-3-carboxamide;N-(3-fluorophenethyl)-2-ethylquinoline-3-carboxamide;N-((furan-2-yl)methyl)furan-2-carboxamide; N-butylfuran-2-carboxamide;N-(4-fluorophenethyl)furan-2-carboxamide;N-(3-fluorophenethyl)furan-2-carboxamide;(furan-2-yl)(2,2,6,6-tetramethylpiperidin-1-yl)methanone;(2,2,6,6-tetramethylpiperidin-1-yl)(perfluorophenyl)methanone;2,3,4,5,6-pentafluoro-N-(5-methylisoxazol-3-yl)benzamide; and (benzo[d][1,3]dioxol-5-yl)(4-(4-fluorophenyl)piperazin-1-yl)methanone.
 3. Amethod of increasing pigmentation in a cell, said method comprising:providing a compound according to claim 1 and contacting a cell with thecompound under conditions effective to induce melanin expression in thecell, thereby increasing pigmentation.
 4. The method according to claim3, wherein the compound is selected from the group ofN-(4-fluorophenethyl)-2-ethylquinoline-3-carboxamide;N-(3-fluorophenethyl)-2-ethylquinoline-3-carboxamide;N-((furan-2-yl)methyl)furan-2-carboxamide; N-butylfuran-2-carboxamide;N-(4-fluorophenethyl)furan-2-carboxamide;N-(3-fluorophenethyl)furan-2-carboxamide;(furan-2-yl)(2,2,6,6-tetramethylpiperidin-1-yl)methanone;(2,2,6,6-tetramethylpiperidin-1-yl)(perfluorophenyl)methanone;2,3,4,5,6-pentafluoro-N-(5-methylisoxazol-3-yl)benzamide; and(benzo[d][1,3]dioxol-5-yl)(4-(4-fluorophenyl)piperazin-1-yl)methanone.5. The method according to claim 3, wherein the cell is in vitro.
 6. Themethod according to claim 3, wherein the cell is in vivo.
 7. The methodaccording to claim 6 further comprising: administering the compound to asubject under conditions effective to cause the compound to contact thecell.
 8. A composition comprising a compound according to claim 1 and acarrier.
 9. The composition according to claim 8, wherein the carrier isselected from the group of lotion, gel, solution, or aerosol.
 10. Thecomposition according to claim 8 in the form of a sunless tanninglotion.
 11. A method of increasing skin pigmentation, said methodcomprising: applying a composition according to claim 8 to skin on asubject in a manner and quantity effective to induce melanin productionin contacted skin cells, thereby increasing skin pigmentation within atreated area of the skin.
 12. The method according to claim 11, whereinthe compound is selected from the group ofN-(4-fluorophenethyl)-2-ethylquinoline-3-carboxamide;N-(3-fluorophenethyl)-2-ethylquinoline-3-carboxamide;N-((furan-2-yl)methyl)furan-2-carboxamide; N-butylfuran-2-carboxamide;N-(4-fluorophenethyl)furan-2-carboxamide;N-(3-fluorophenethyl)furan-2-carboxamide;(furan-2-yl)(2,2,6,6-tetramethylpiperidin-1-yl)methanone;(2,2,6,6-tetramethylpiperidin-1-yl)(perfluorophenyl)methanone;2,3,4,5,6-pentafluoro-N-(5-methylisoxazol-3-yl)benzamide; and (benzo[d][1,3]dioxol-5-yl)(4-(4-fluorophenyl)piperazin-1-ylmethanone. 13.-20.(canceled)